Summary

One of the most significant problems facing developmental biologists who do
not work on an organism with well-developed genetics - and even for some who
do - is how to inhibit the action of a gene of interest during development so
as to learn about its normal biological function. A widely adopted approach is
to use antisense technologies, and especially morpholino antisense
oligonucleotides. In this article, we review the use of such reagents and
present examples of how they have provided insights into developmental
mechanisms. We also discuss how the use of morpholinos can lead to misleading
results, including off-target effects, and we suggest controls that will allow
researchers to interpret morpholino experiments correctly.

Introduction

To understand the molecular basis of early development, developmental
biologists have long wished for techniques that allow the experimenter
specifically to inhibit the functions of particular genes in particular cells
at particular developmental stages. That goal has not yet been achieved,
although researchers working on mouse embryos are closest to this aim, with
their use of targeted mutations and Cre recombinase. Even here, however, there
are some difficulties: attempts to interfere with the function of one gene may
lead to unwanted side-effects on another
(Carvajal et al., 2001), and
users of Cre recombinase need to be aware of the potential toxicity of Cre
expression (Schmidt-Supprian and Rajewsky,
2007).

But what of other species? There is no doubt that research on other
vertebrates and on invertebrates has led to fundamental insights into early
development, and that the use of such species can have significant practical
advantages over the use of mammalian embryos, in terms of accessibility, cost
and time, in addition to their intrinsic interest. In none of these species is
it yet possible routinely to carry out specific gene targeting, and although
conventional genetic screens are of inestimable value in understanding a
particular process, they cannot be guaranteed to target the desired gene nor
can they be guaranteed to produce a null mutation. Thus, researchers need a
way to inhibit the functions of genes. Dominant-negative approaches have their
place, but the best hope, certainly in terms of specificity, lies in antisense
RNA technologies. Antisense RNA strategies can be applied not only to
`genetic' invertebrate and vertebrate organisms, but also to `non-genetic'
organisms and to tissue culture cells, and they may be of increasing use in
searches for new drugs.

Both RNA interference (RNAi) and morpholino (MO) techniques have had a huge
influence on our understanding of gene function in the early embryo, and will
continue to do so. But are these techniques too good to be true? All antisense
techniques, with the possible exception of those employed by the animal
itself, suffer from some off-target effects. These effects can make it
difficult to discern how much of an antisense-mediated phenotype results from
the knockdown of the gene of interest, and how much results from the knockdown
of other genes. Clarifying this issue requires the establishment of stringent
controls that are applied across the board. Here, we discuss appropriate
controls for antisense technologies for the study of animal development,
focussing on controls for MO experiments, and we make recommendations about
the use of these controls in zebrafish and frog embryos. These issues have
been discussed elsewhere for RNAi experiments in the fruit fly Drosophila
melanogaster (Dasgupta et al.,
2007; Ma et al.,
2006) and in mammalian cells
(Svoboda, 2007). Some of the
controls we recommend are also likely to be important for establishing the use
of MOs for other species and for therapeutic applications.

Antisense techniques

The idea of introducing antisense RNA into a cell to inhibit the
translation, processing or stability of its endogenous mRNA complement was
developed over 20 years ago (Izant and
Weintraub, 1984; Izant and
Weintraub, 1985). The technique proved successful in inhibiting
the translation of exogenous RNA injected into oocytes of the frog Xenopus
laevis (Harland and Weintraub,
1985; Melton,
1985), as well as in inhibiting the translation of endogenous mRNA
(Izant and Weintraub, 1985).
And the use of antisense oligonucleotides in the hands of Heasman and Wylie
(Wylie and Heasman, 1997) has
allowed researchers to study the functions of maternally inherited transcripts
in frog oocytes, including those encoded by genes such as Vg1 and
VegT (Birsoy et al.,
2006; Zhang et al.,
1998). In these cases, the antisense reagent interfered with gene
function by hybridising to endogenous RNAs and by mediating their degradation
via RNaseH (Summerton, 1999).
Further studies in the nematode worm Caenorhabditis elegans revealed,
surprisingly, that double-stranded (ds) RNAs also interfere with endogenous
gene function (Fire et al.,
1998). This work led to the discovery in animals of families of
small, non-coding RNAs that regulate many, if not all, aspects of gene
expression (Farazi et al.,
2008; Mendes Soares and
Valcarcel, 2006), and to a Nobel prize for the discoverers of
dsRNA-mediated interference (Couzin,
2006).

At least in the frog, however, with just a few exceptions
(Li and Rohrer, 2006;
Lombardo and Slack, 1997;
Nakano et al., 2000;
Steinbeisser et al., 1995),
the use of conventional antisense RNA, antisense oligonucleotides, or RNAi has
not met with success in understanding the roles of zygotically expressed genes
(Heasman, 2002). Similarly, in
the zebrafish Danio rerio, antisense RNA has been shown to have
widespread effects that are sequence independent, preventing the practical
application of this approach to the study of specific gene functions during
development (Oates et al.,
2000). It remains possible that new approaches will make
techniques such as RNAi feasible in these species, but for now they do not
seem to be practicable.

These problems in testing gene function delayed the field until early this
century, when it was shown, with some fanfare [Editorial, Nature
Genetics 26(2), 129-130], that MOs could be targeted to knockdown
specific gene expression both in frog
(Heasman et al., 2000) and in
zebrafish (Ekker, 2000;
Nasevicius and Ekker, 2000).
The power of MOs to test gene function was quickly recognised and applied to
other organisms, including another species of frog, X. tropicalis
(Nutt et al., 2001), the chick
Gallus gallus (Kos et al.,
2001), an ascidian, Ciona savignyi
(Satou et al., 2001), oocytes
of the mouse Mus musculus
(Coonrod et al., 2001), and
the sea urchin, Strongylocentrotus purpuratus
(Coffman et al., 2004). The
importance of MO technology in advancing the field was heralded by an entire
issue of the journal Genesis [30(3), July 2001], devoted to gene
targeting studies employing MOs in a variety of organisms.

Structures of conventional DNA and morpholino oligonucleotides.
(A) Conventional DNA oligonucleotide. (B) Morpholino
oligonucleotide. Note the six-membered morpholino ring in B and the non-ionic
phosphorodiamidate link between the two rings.

MOs are synthetic oligonucleotides composed of chains of about 25 subunits
that are similar to DNA and RNA oligonucleotides, except that they have a
morpholine ring rather than a ribose ring
(Fig. 1). This feature still
allows MOs to undergo Watson-Crick base pairing, but it offers significant
advantages over conventional oligonucleotides
(Corey and Abrams, 2001;
Heasman, 2002;
Heasman et al., 2000). In
particular, MOs are resistant to nucleases and are therefore remarkably stable
(see below), and the fact that they do not carry a negatively charged backbone
means that they are less likely to interact non-specifically with other
components of the cell and may be less toxic as a result.

MOs do not act through an RNaseH mechanism but instead can be designed to
inhibit translation (Summerton,
1999) (Fig. 2A),
or, as more recent experiments have shown, to prevent the proper splicing of
RNA (Draper et al., 2001)
(Fig. 2B-E). As we discuss in
this article, MOs are now used in a wide variety of applications. For example,
a recent study shows that it is possible in principle to use MOs to do the
same types of genome-wide screens in vertebrate embryos, such as X.
tropicalis (Rana et al.,
2006), that have been done successfully using RNAi in C.
elegans embryos (Gonczy et al.,
2000; Kamath et al.,
2003; Sonnichsen et al.,
2005) and in tissue culture cells in D. melanogaster
(Agaisse et al., 2005;
Boutros et al., 2004;
Friedman and Perrimon, 2006).
This study (Rana et al., 2006)
also showed that fluorescent MOs can be used to monitor the distribution of
MOs within the embryo. More recently, photoactivatable MOs have been used to
provide spatiotemporal control over gene knockdown, raising the possibility of
targeting gene perturbations to specific regions of the embryo, perhaps even
in individual cells, at particularly relevant stages of development
(Shestopalov et al., 2007)
(Fig. 2F). Finally, MOs can
also be used to block the functions of microRNAs, one of the non-coding RNA
families in animals identified as a result of the original dsRNA study. This
can be achieved by targeting either the mature microRNA or the microRNA
precursor (Kloosterman et al.,
2007).

Use of morpholinos in frogs, zebrafish and other organisms

The first descriptions of the use of MOs in developmental biology research
were made eight years ago (Ekker,
2000; Heasman et al.,
2000; Nasevicius and Ekker,
2000), and since then the research community has adopted them with
enthusiasm. The ease of use of MOs, especially in zebrafish and frogs, and the
exciting results that have been obtained, have meant that any paper describing
a new gene cannot afford to neglect experiments using an MO to knockdown the
gene and thus to learn about its normal function. However, an unfortunate side
effect of the enthusiasm for MOs is that because proper standards for controls
have not been adopted, there are undoubtedly publications in which at least
some of the supposedly specific results are due to off-target effects, rather
than to the specific knockdown of only the gene(s) of interest. One purpose of
this review is to discuss this issue and to suggest a series of standard
controls; our suggestions complement and supplement those put forward by Corey
and Abrams (Corey and Abrams,
2001).

Translation-blocking MOs

Many investigators use MOs directed against the initiation codon of the
target mRNA. This approach is particularly attractive in those (now rather
few) model species whose genome sequence is unknown, because one does not need
to know the intron-exon structure of the target gene to inhibit its function.
The design of MOs intended to inhibit translation has been extensively
studied, and the rules for success prove to be relatively simple (see
Box 1).

There are so many examples of the use of translation-blocking MOs that it
is difficult to select just a few to describe. Here, we mention two examples,
one from each of our labs. Both cases were very informative about the function
of specific proteins during development. The first example describes a
straightforward case, whereas the second example describes a case in which
detective work was necessary to solve a mystery.

In the first example, MOs were used to explore the role of Notch signalling
in zebrafish neurogenesis. In the absence of Notch signalling, cells that
would normally become trunk neural crest develop as supernumerary Rohon-Beard
sensory neurons (Cornell and Eisen,
2000), suggesting that Notch signalling is required for the neural
crest fate. However, the use of MOs to knockdown Neurogenin 1 (Neurog1), a
proneural protein, revealed that Notch signalling is required only to prevent
neurogenesis, because the absence of both Notch signalling and Neurog1
restored normal trunk neural crest formation
(Cornell and Eisen, 2002).

The MO should normally be about 25 bases in length, with a GC content of
about 50%, and little or no secondary structure.

The MO should be designed to be complementary to sequence between the
5′ cap and about 25 bases 3′ of the AUG translation start site.
There is a sharp decrease in the efficacy of MOs that are positioned any more
3′ of the translation start site than this.

It is wise to carry out a BLAST search using the proposed sequence to
confirm that it, or a similar sequence, is not present elsewhere in the genome
of the species under study.

It is also wise to re-sequence the MO target in the particular strain under
study, to avoid being misled by SNPs or sequencing errors.

MOs should be stored in aliquots at -20°C. They can be heated to
65°C and then put on ice prior to injection. The concentration of a
dissolved MO can be calculated by measuring its absorbance at 265 nm in 0.1 N
HCl and dividing by ϵ, the molar absorbance provided on the MO product
information sheet (see
www.gene-tools.com).

In the second example, MOs were used to investigate the function of activin
in the early X. laevis embryo. Activin had long been known to have
powerful mesoderm-inducing activity in amphibian embryos
(Albano et al., 1990;
Asashima et al., 1990;
Smith et al., 1990;
Thomsen et al., 1990), but its
role in normal development was unclear until MOs were used to inhibit its
effects (Piepenburg et al.,
2004). In the course of these experiments, a MO designed to
inhibit translation of activin B caused defects in development, as well as the
dose-dependent downregulation of several mesodermal markers. A control MO with
four evenly spaced nucleotide changes had no such effect, suggesting that the
phenotype was specific. But, to the authors' surprise, no phenotype was
observed with a second MO, positioned 5′ of the first, which was
predicted to be equally effective in inhibiting activin B translation.
Eventually, it was found that the 5′ untranslated region of activin B
derived from the X. laevis colony maintained in the authors'
laboratory differed from that of the published sequence, such that two
nucleotides in the second MO did not match the target. When a perfectly
matched MO was injected into X. laevis embryos, it recapitulated the
original phenotype, indicating (but not proving) that the effect is specific.
`Rescue' experiments (see below) also indicated that the MOs used exerted a
specific effect, and the authors went on in later experiments to demonstrate
that the loss of activin function causes the misregulation of genes involved
in cell-cycle regulation (Ramis et al.,
2007). In addition to providing new insights into the role of
activin B, these experiments make the point that it is important to
re-sequence the MO target sequence in the strain of animal under study, to
avoid being misled by single-nucleotide polymorphisms (SNPs) or errors in
published sequences.

Different uses of antisense morpholino oligonucleotides. (A)
Inhibition of translation by an antisense morpholino oligonucleotide. The MO
is targeted to sequence 5′ of the translation start site and inhibits
progression of the initiation complex. (B-E) Use of MOs to prevent
correct splicing of X. tropicalis NRH1. (B) The first three exons of
NRH1. Arrows indicate the positions of the forward (F1)
and reverse (R1, R2) PCR primers used to test the
efficacy of the splicing MOs. Exon 1 is 326 bp, intron 1 is 9013 bp, exon 2 is
134 bp, intron 2 is 740 bp, and exon 3 is 363 bp. If the mRNA is correctly
spliced, the combination of F1 and R1 should yield no
PCR product and F1 and R2 should give a product of 490
bp. (C) The +intron1 MO is designed to cause intron 1 to be retained. This
would result in the first 15 amino acids of NRH1 being followed by 67 missense
amino acids before a stop codon is reached. If intron 1 is retained in this
way, the combination of F1 and R1 should give a PCR
product of 280 bp, and F1 and R2 should yield a product
in excess of 9.0 kb. (D) The Δexon2 MO is designed to exclude exon 2
from the mature RNA. In this case, exon 3 would no longer be in frame with
exon 1, and a truncated protein consisting of 15 correct amino acids followed
by 25 missense residues would be formed before a stop codon is encountered.
Deletion of exon 2 in this way should cause the combination of F1
and R1 to yield no PCR product, and F1 and R2
should yield a product of 360 bp. (E) Verification of the efficacy of MOs
+intron1 and Δexon2. Comparisons of lane 2 with lane 3, and lane 6 with
lane 7, indicate that both MOs reduce levels of correctly spliced transcript
by ≥50%. Note that the predicted 9.5 kb band in lane 4 is absent, probably
because the PCR conditions do not efficiently amplify products of this size.
(F) Activation of a caged morpholino. An inhibitory oligonucleotide is
coupled to a specific MO through a photocleavable linker. Irradiation [10
seconds of 360 nm light (hν)] cleaves the linker and allows the MO to exert
its effects. B-D are redrawn, and E is reproduced, with permission, from Knapp
et al. (Knapp et al., 2006). F
is redrawn, with permission, from Shestopalov et al.
(Shestopalov et al.,
2007).

Splice-inhibiting MOs

The difficulty of ensuring that MOs designed to inhibit translation do
actually knockdown protein levels and thus have their intended effect (see
below) caused researchers to investigate the possibility of using MOs to
inhibit pre-mRNA splicing or even to cause exon skipping
(Draper et al., 2001;
Gebski et al., 2003).
Splice-inhibiting MOs have the advantage that one can quantify the efficacy of
the MO (Draper et al., 2001).
In addition, because such MOs do not affect spliced maternal transcripts they
will normally allow genetic mutations to be phenocopied when the gene in
question also has a maternal function
(Bennett et al., 2007;
Gore et al., 2007;
Gore et al., 2005). MOs
designed to inhibit splicing have often been used to corroborate data obtained
with MOs that block translation. As with translation-blocking MOs, there are
now many examples of the use of splice-inhibiting MOs. Thus, we have chosen
two examples, one from each of our labs. In the first example, the Eisen
laboratory showed that the knockdown of zebrafish Islet1 protein with two
different splice-inhibiting MOs, either individually or in combination,
results in the same phenotype. In this case, motoneurons in MO-injected
embryos develop interneuron-like characteristics, providing evidence that
Islet1 functions both to promote motoneuron differentiation and to inhibit the
differentiation of some ventral interneurons
(Hutchinson and Eisen,
2006).

In the second example, the Smith group investigated the function of
neurotrophin receptor homologue (NRH1) proteins in mesoderm formation in frog
embryos (Knapp et al., 2006).
MOs targeted against the translation start sites of two related X.
laevis proteins indicated that NRH1 activity is required for expression
of Xbra and Chordin in the early embryo, but that
Goosecoid expression is less dependent on signalling through this
receptor. The downregulation of Xbra and Chordin was also
observed in X. tropicalis (a diploid species whose genome has been
sequenced), using one MO designed to inhibit the translation of NRH1, and two
MOs that prevented its correct splicing. The fact that similar results were
obtained in two species, and using both translation-blocking and
splice-inhibiting MOs, suggests that the observed effects are specific.

There has been considerable work on the best design for splice-inhibiting
MOs (Morcos, 2007). In
contrast to MOs designed to block translation, it is not possible to apply
general guidelines to the design of splice-inhibiting MOs, because genes
differ in their genomic structures and proteins in their functional domains.
For example, a truncated version of a protein may act as a dominant-negative
or even as a constitutively active form of the wild-type protein, or retention
of an intron might, if the mRNA remains in frame, not affect the function of a
protein. It is also possible that the use of splice-inhibiting MOs will reveal
cryptic splice donor or acceptor sites, resulting in the retention of a short
stretch of intron or the deletion of a short stretch of exon. This might cause
in-frame insertions or deletions in the protein of interest. The important
point, therefore, is to determine the sequence of the modified mRNA, to ask
what protein is encoded by it, and, if necessary, to test the function of that
protein.

microRNA-blocking MOs

MicroRNAs are short (about 22 nucleotides), non-coding RNA molecules that
regulate gene expression by both blocking the translation and promoting the
decay of their target mRNAs (Alvarez-Garcia
and Miska, 2005; Bushati and
Cohen, 2007; Farazi et al.,
2008). Various approaches have been used to block the functions of
individual microRNAs by means of antisense technologies. The most direct has
been to use antisense oligonucleotides that are complementary to the target
microRNA, and for these oligonucleotides to be modified in some way to
increase their stability. These modifications include the introduction of
2′-O-Me-modified nucleotides and phosphorothioate linkages;
and, to assist their delivery into liver cells of living mice, such 23-mer
RNAs have been covalently linked to cholesterol molecules
(Krutzfeldt et al., 2006;
Krutzfeldt et al., 2005).
Recently, MOs have also been used in such an approach. For example, in
zebrafish, MOs complementary to microRNA-214 (miR-214) have
revealed that this microRNA regulates expression of su(fu), which
encodes a modulator of Hedgehog signalling
(Flynt et al., 2007), and MOs
complementary to miR-140 have revealed that this microRNA regulates
Pdgf signalling during palate formation
(Eberhart et al., 2008). In
X. laevis, the activities of miR-15 and miR-16 have
been inhibited by the use of 2′-O-methylantisense
oligonucleotides and of MOs to reveal that these microRNAs target the type II
Nodal receptor Acvr2a (Martello et al.,
2007).

In an intriguing alternative approach, Schier and his colleagues have
`protected' the two zebrafish miR-430 targets ndr1 (also
known as squint; this gene encodes a Nodal-related signalling
molecule) and lefty (a Nodal antagonist) by introducing `target
protectors', MOs that are complementary to the microRNA binding sites.
Protection of the ndr1 miR-430 binding site resulted in an elevation
of Nodal signalling, and protection of the lefty site caused a
diminution (Choi et al.,
2007). This result illustrates both the utility of MOs in
endeavours of this sort, as well as the roles of microRNAs in modulating
levels of Nodal signalling.

Problems with MOs

For all these successes, there are difficulties with the MO technique, and
there have undoubtedly been some misleading results. The main difficulties in
interpreting experiments fall into three classes (see
Box 2). First, it is important
to know how effective the knock-down has been, and, second, the possibility of
`off-target' effects has to be addressed; that is, the possibility that the MO
inhibits the function of an irrelevant gene instead of, or in addition to, the
intended gene. As discussed below, in addressing these problems, it is very
helpful if the genome of the species under investigation has been sequenced
and well annotated. The third problem is that it can be difficult, especially
in small embryos, to inject precise and reproducible volumes of MO.

It is also important in interpreting experiments that make use of antisense
oligonucleotides to consider the mechanisms by which the reagent of choice
acts. For example, RNAi leads to the destruction of mRNA by RNase H activity,
whereas MOs interfere only with translation or with pre-mRNA splicing. In all
cases, if the experiment is designed properly, the protein that is encoded by
the mRNA should be knocked down by the antisense reagent. However, the mRNA
may or may not persist, depending on the antisense strategy that is used, and,
in some cases, mRNA levels may even increase, as the embryo attempts to
compensate for loss of the protein. In many cases, this may be of no
importance, but if the RNA has functions instead of, or in addition to, its
role as mRNA, changes in its level may be very significant indeed. For
example, the knockdown of VegT mRNA causes a decrease in VegT
protein, but it also causes the release of Vg1 mRNA from the vegetal
cortex of X. laevis embryos and a decrease in the amount of Vg1
protein (Heasman et al.,
2001). Bicaudal C and Wnt11 mRNAs also become
dispersed from the vegetal cortex. This phenomenon is observed only if
VegT mRNA is destroyed; MOs designed to inhibit VegT translation but
not to degrade VegT RNA have no effect on RNA localisation
(Heasman et al., 2001). In
this particular case, `rescue' experiments, in which VegT mRNA is
injected into the oocyte in an effort to replace the depleted transcripts,
suggest that the release of Vg1, Bicaudal C and Wnt11 mRNAs
from their vegetal position does not compromise development significantly,
because such embryos still form all three germ layers. In other cases,
however, the phenotype of embryos injected with a particular antisense RNA or
MO may indeed depend on whether or not the target mRNA persists.

Box 2. Three potential problems with the use of MOs

It can be very difficult to estimate the efficacy of MOs without a good
antibody.

It is very difficult to rule out the possibility that the MO inhibits the
function of an irrelevant gene, as well as, or instead of, its intended
target.

Especially in smaller embryos, it can be very difficult to inject precise
and reproducible volumes of MOs.

Effectiveness of gene knockdown

As discussed above, many investigators use MOs targeted against the
initiation codon of the target mRNA. Simple as this approach might be, it
suffers from the fact that it is not possible to determine, without a suitable
way to test for the protein, how effectively the protein has been knocked
down. If an antibody is available that works well in vivo, it can provide a
high-resolution view of protein expression, even at the single-cell level, and
such antibodies can reliably report the downregulation of protein in cells of
MO-injected embryos (Hutchinson and Eisen,
2006). However, the antibody might not work well in vivo, in which
case it might be necessary to use western blotting. Unfortunately, a
significant drawback of this technique is that the sensitivity might be such
that one can determine effectiveness only on a rather large population of
animals. Similarly, although it might be possible to test for the presence of
a protein using an enzyme assay or a protein gel, these approaches are likely
to require a large number of animals and may not give the resolution required
for the experiment.

The determination of knockdown efficiency is clearly an essential step. A
common alternative is to express a form of the target protein that is tagged,
for example with the influenza haemagglutinin (HA) epitope or green
fluorescent protein (GFP), and to show that the MO can knockdown expression of
the tag in question (Collart et al.,
2005). Such an experiment is better than nothing - it demonstrates
at least that the MO can recognise its target mRNA in the cytoplasmic milieu -
but it cannot confirm that the endogenous protein is targeted, nor can it
predict the extent to which expression of the endogenous protein is inhibited.
It is therefore of the greatest importance to obtain a high-affinity specific
antibody for the gene product under study. This might prove to be difficult
and expensive, but there is no doubt that such an antibody will be an
essential research tool.

MOs designed to inhibit splicing have the great advantage that one can
determine, by PCR (polymerase chain reaction), how effective the reagent has
been, although it is important to note that a 50% reduction in mRNA levels
does not necessarily lead to a 50% reduction in protein levels! However,
incorrectly spliced pre-mRNAs often remain in the nucleus and their nuclear
localisation can be visualised using RNA in situ hybridisation
(Hutchinson and Eisen, 2006),
providing another measure of MO efficacy.

In the case of MOs designed to knockdown particular microRNAs, their
efficiency can be estimated by northern blots or by RNA in situ hybridisation.
For example, Plasterk and colleagues have investigated the function of
miR-375 in zebrafish and shown that this microRNA is necessary for
normal formation of the pancreatic islet
(Kloosterman et al., 2007). In
the course of control experiments, an MO complementary to mature
miR-206 was injected into zebrafish embryos and, at intervals
thereafter, embryos were subjected to RNA in situ hybridisation and northern
blotting. Both techniques revealed a significant reduction in levels of
miR-206 that was specific in the sense that levels of another
microRNA, miR-124, were unaffected by the miR-206 MO.
Additional experiments indicated that this decrease in miR-206 levels
did not occur because the MO interferes with the RNA isolation procedure, nor
because MOs affect microRNA stability. Rather, the MOs inhibit microRNA
maturation. Consistent with this suggestion, MOs targeting the cleavage sites
of the Drosha and Dicer enzymes that process precursor microRNAs also caused a
loss of miR-206 (Kloosterman et
al., 2007).

Although MOs are remarkably stable
(Nutt et al., 2001;
Summerton, 2007), their levels
may nevertheless fall slightly as development proceeds, and, although
zebrafish and frog embryos do not increase in volume during development,
numbers of nuclei increase dramatically, so that the intranuclear
concentration of MOs designed to inhibit splicing may decrease significantly.
Thus, it cannot be assumed that a MO that is effective for the first few hours
or days following injection will remain so over a longer period of time. It is
therefore essential to assay the efficacy of MOs over the entire time course
of an experiment. If the efficacy of a particular MO is inadequate at later
stages of development, it may be possible to use iontophoresis
(McWhorter et al., 2003) or
electroporation (Thummel et al.,
2006) into individual or small groups of cells to supplement MO
levels.

Off-target effects of MOs

A more insidious problem with the use of MOs, including those targeted to
the initiation codon and those designed to inhibit splicing, is that they may
have `off-target' effects. In other words, they might affect the production of
a completely irrelevant gene product, and the observed phenotype may be only
partially the result of, or in the worst case have nothing to do with, the
gene under study. Two published examples of such off-target effects come from
work on sea urchins and zebrafish. Unfortunately, there are likely to be many
other cases, many yet to be recognised, in which phenotypes caused by MOs are
non-specific.

In the sea urchin example, embryos injected with either of two MOs directed
against the S. purpuratus Runt gene, SpRunt, caused early
developmental arrest, whereas injection of either of two other MOs produced
gastrula stage defects (Coffman et al.,
2004). The first two MOs were the more effective at reducing
levels of SpRunt protein, but, remarkably, they also targeted two different
histones, thus causing the early phenotype. One MO, designated m1 and designed
to inhibit the translation of SpRunt, inhibited the synthesis of histone H3,
whereas another, designated m4 and designed to interfere with the splicing of
SpRunt mRNA, inhibited the translation of histone H4. It is to the
credit of the authors that they carried out meticulous controls in their
experiments (see below), and it is sobering to realise that in an MO of 25
nucleotides, just 18 (in the case of m1) or 20 (in the case of m4) need match
a target sequence for inhibition of protein synthesis to occur. As the authors
point out, S. purpuratus embryos develop at 15°C, so the
`stringency' of hybridisation is lower in sea urchins than in some other
species, but the point is clear nevertheless. We note that X. laevis
embryos can be cultured between 14°C and 24°C. Thus, until someone
carries out an experiment to directly test the effectiveness of the same MO in
embryos raised at different temperatures, to circumvent the potential problem
of the relationship between `stringency' and temperature, it may be sensible
for embryos injected with MOs to be incubated closer to the higher
temperature.

In the zebrafish example, researchers in Julian Lewis's laboratory were
studying the interaction between the membrane-associated scaffolding protein
Magi1 and the Notch ligand DeltaD (Wright
et al., 2004). Two MOs, one designed to interfere with splicing of
the magi1 mRNA and one directed against its translation start site,
caused a narrowing of the hindbrain and midbrain of the embryo. This phenotype
appeared specific, because it was not observed when the researchers used a
version of the translation-blocking MO that had five nucleotide changes.
However, this phenotype had been observed previously as a non-specific effect
of MOs (Ekker and Larson,
2001), and `spurred on by the doubts of a [Development]
referee', the authors went on to demonstrate that deltaD mutant
embryos did not show the narrowed-ventricle phenotype, but that it did occur
when these embryos were injected with the Magi1 MOs described above. Thus, it
appears that the Magi1 MOs cause a phenotype that is robust and consistent,
but nevertheless non-specific.

Box 3. A summary of our recommendations

Comparison with existing mutant. Should a mutation in the gene of
interest be available, a careful comparison should be made between the
phenotype of the mutant and that of morpholino-injected embryos, so as to
reveal any off-target morpholino effects. Loss of protein. Whenever
possible, the effect of each morpholino on protein levels should be verified
by antibody staining or other assays.

Incorrectly spliced pre-mRNA. A splice-inhibiting morpholino should
be used for every target gene that has more than one exon. The effect of
splice-inhibiting morpholinos should be verified by RT-PCR; the altered splice
products should be verified by sequencing, and where necessary the function of
the modified protein thus encoded should be investigated. In addition, RNA in
situ hybridisation should be used to determine whether unspliced pre-mRNA is
retained in the nucleus.

Minimisation of off-target effects. At least two morpholinos should
be designed against each target gene; it is best to have both a
translation-blocker and a splice-inhibitor. These morpholinos should be tested
independently to ensure that they give similar phenotypes, and also
simultaneously to test for synergism. The latter will involve titrating these
morpholinos down to a level at which they yield no phenotype, co-injecting
them into embryos, and showing that together they yield the same phenotype as
they yield independently. It is important to ensure that injection volumes are
consistent in such experiments.

RNA rescue. Co-injection of a form of the targeted RNA that is not
recognised by the morpholino should be used to show that the effects of the
morpholino are specific. In the case of morpholinos designed to interfere with
splicing, the rescuing RNA can be the wild-type version; in the case of
morpholinos designed to interfere with translation, the rescuing RNA should be
made `immune' to the morpholino in question, either by removing the
appropriate regions of the 5′ untranslated region or by creating silent
mutations in the coding sequence.

Control morpholino. A control morpholino should always be used. This
could be the standard control morpholino, a morpholino that affects a gene
that is not expressed in the cells of interest, or a mismatch, scrambled or
invert morpholino. We favour a five-base mismatch morpholino because this is
most similar to the experimental morpholino, but one or more of the other
control morpholinos may be more appropriate under some conditions.

The non-specific neural defects observed in the above experiments, and in
15-20% of all MO experiments carried out in zebrafish, are likely, as
explained below, to derive from the activation of p53 and consequent
p53-induced apoptosis (Robu et al.,
2007). The mechanism by which some MOs activate p53 is unknown,
and it is also a mystery that MOs activate predominantly a truncated form of
p53 (the D113 isoform), whereas it is the full-length version that is required
for the induction of cell death. Nevertheless, a MO that targets the
translation start site of p53 is effective at relieving non-specific neural
cell death in response to injection of some MOs
(Robu et al., 2007), and this
is likely to prove a powerful adjunct to MO experiments in the zebrafish
embryo. The caveat here is that it may be difficult to use MOs to study
developmental processes that involve p53-mediated cell death.

It is also worth noting that not all cell death in experiments using MOs is
non-specific. For example, a reduction in Cytochrome C oxidase activity caused
by MO injection results in cell death in the hindbrain and neural tube, but
this cannot be reduced by co-injection of a p53 MO
(Baden et al., 2007).

These examples should suffice to illustrate the potential hazards of using
MOs in the study of early development. To what extent can use of the right
controls allow researchers to distinguish between bona fide and spurious
results?

Appropriate controls

Given the potential problems described above, how can investigators ensure
that the phenotype they observe is specific, that it is the genuine
consequence of the loss of function of the gene under investigation? We
suggest several approaches and we recommend that they become standard controls
for the use of MOs (see Box 3).
We recognise that some of the controls we recommend will not be appropriate or
will not work in every case. However, we suggest that researchers use as many
controls as possible, so as to provide the strongest evidence for MO
specificity.

The right control construct

The most obvious strategy to control MO experiments is to create control
animals that are identical to the MO-injected experimental animals in every
respect except that the MO injected into the controls should not interfere
with the function of the gene being tested. One type of control is a MO that
targets an exogenous gene that is not present in the species being tested. For
example, the company Gene Tools provides a `Standard Control' MO directed
against human β-globin pre-mRNA
(www.gene-tools.com/node/23#standardcontrols).
Although this reagent controls for the use of MOs in general, it does not
necessarily control for the use of any specific MO, and in particular it does
not control for the activation of p53 (see above). Therefore, for some
experiments (for example, those where it is not possible to carry out a
`rescue'), it might be preferable to select as a control a MO that more
closely resembles the experimental MO. This control MO might be a randomised
version of the specific MO, or its invert, or it might differ in a particular
number of nucleotides. Unfortunately, there is no consensus on how many
nucleotides should differ in the control MO, and the number may not be the
same for different target sequences (depending, for example, on how GC-rich
they are, on the species under study, or on the culture conditions). As
discussed above, it is possible that MOs introduced into species that develop
at a lower temperature might recognise their targets even if several
nucleotides have been changed.

Our experience has shown that a 25-mer MO that differs in four nucleotides
from the experimental MO can have an effect similar to the experimental MO
when injected at high concentration into zebrafish embryos, but no effect when
injected at a lower concentration (Cornell
and Eisen, 2002). Similarly, in X. tropicalis, 25-mer MOs
that differ in five nucleotides from the target sequence have little effect on
translation (Rana et al.,
2006). Thus, based on this rather limited information, if the Gene
Tools standard control MO is inappropriate, we recommend using five-nucleotide
mismatch MOs as a control for the injection process itself, for the
introduction of significant amounts of exogenous oligonucleotides, and for the
specificity of a particular MO. To our knowledge, no one has published a
systematic comparison of the optimal number of nucleotide mismatches under
various conditions, and although such a study would be expensive to undertake,
it would provide important baseline information for the design of MO controls.
In the meantime, in designing MOs, it would be wise to conduct a BLAST search
using the proposed sequence, and to reject the sequence if it differs by fewer
than five nucleotides from a sequence near the translation start site of any
unrelated gene or indeed near a splice junction of an unrelated gene.

Use of mismatch MOs

We described above that MOs that differ by five out of 25 nucleotides from
their target sequence appear not to interfere with translation of the target
mRNA, whereas at least some MOs that differ from the target sequence by four
nucleotides do interfere, at least when injected at high concentration. It is
instructive, and sobering, to ask how likely it is that a 25-mer might
recognise a target that differs from the intended one. Theoretical
calculations based on the X. tropicalis genome sequence indicate that
there is a probability of about 0.3 that a particular translation-blocking MO
will match another target sequence near a translation start site with four or
fewer mismatches (Rana et al.,
2006). And if one takes into account the possibility that the MO
might instead interfere with mRNA splicing, the chance of off-target effects
increases to 0.5. The strength of a phenotype obtained using a MO with three
or four mismatches is likely to be relatively weak, but clearly, even if a
`control' MO causes no phenotype, it is still possible that the phenotype
observed with the `specific' MO is an artefact. Therefore, although using a
five-nucleotide mismatch MO control is wise, by itself this control is
insufficient, and we recommend using additional controls, as described below.
We note that, although these calculations were made for X.
tropicalis, the conclusions will be similar for vertebrate species of
comparable genome sizes.

Use of different MOs targeting the same gene

The pessimistic calculations outlined above suggest that for every two MOs
introduced into a developing embryo, one will elicit non-specific effects. At
first sight this represents an untenable situation: how can such experiments
be interpreted? But in practice things are not so bad. The strengths of the
off-target phenotypes are likely to be weak, and concerns can be addressed by
designing a second, non-overlapping MO against the gene of interest. The
chance of both MOs yielding an off-target effect may be 0.25, but the chance
of the two off-target effects being the same is considerably lower. Thus, if
the phenotypes caused by the two reagents are similar, one can be more
confident that they really shed light on the function of the intended target
gene. Even more confidence can be gained if the same phenotype is observed
when two MOs are injected together at low levels that do not elicit phenotypes
on their own. This can be achieved by generating a `dose-response' curve for
each MO, by titrating it down to a level at which it does not elicit a
phenotype, and then injecting the two MOs together at these low concentrations
(Maves et al., 2002). In
carrying out such experiments, it is essential to take great care in
controlling injection volumes and to repeat the experiment on at least three
independent occasions. These precautions are crucial because a two- or
threefold variation in injection volumes in quantitative experiments of this
sort may lead to misleading results.

In practice, as shown by the SpRunt experiments described above,
it might be sensible to use three or even four MOs targeted against the gene
of interest. Two of these might be designed to block translation of the
protein and two designed to inhibit correct pre-mRNA splicing. The
interpretation of such experiments must take into account that different MOs
might have different `penetrances', such that a high dose of one MO yields the
same phenotype as a low dose of another. Thus, the experimenter will need to
construct a dose-response curve and inject a range of concentrations of each
MO to make valid comparisons.

Finally, as an alternative to using different MOs, it may be possible to
use closely related species to verify the phenotype produced by a MO. For
example, the function of genes such as NRH1 can be tested in both X.
laevis and X. tropicalis
(Knapp et al., 2006). Such an
approach requires caution, however, and it may be dangerous to make
comparisons across different genera. For example, Fgf8 is necessary
for heart development in ray-fin fish and in tetrapods, but in zebrafish it is
fgf8a that plays this role and in the stickleback Gasterosteus
aculeatus it is fgf8b
(Jovelin et al., 2007). This
observation suggests that the heart function of the two fgf8
duplicates was conserved in the common ancestor of zebrafish and stickleback
after genome duplication in the ray-fin fish lineage, but that the expression
of the two paralogues subsequently diverged. It will be interesting in the
future to design MOs to target the different paralogues of the tetraploid
species X. laevis, to investigate whether they have different
functions, and to compare the resulting phenotypes with those observed in the
diploid species X. tropicalis.

Together, these considerations lead us to suggest that, for any gene, at
least two MOs should be designed, with one, if possible, designed to inhibit
correct pre-mRNA splicing. Even if antibodies are unavailable, this will allow
some quantification of the effect of the MO. We suggest that for each MO a
dose-response curve is prepared, and that co-injection of two MOs at below
threshold levels should also be carried out in an effort to search for synergy
(see Box 3).

Rescue

None of the controls described above is perfect. It is always possible that
a MO will have off-target effects, and it will not always be feasible to
alleviate these by injection, for example, of a MO targeted against p53. The
most reliable control, therefore, is to attempt to `rescue' the phenotype by
introducing the gene product of interest in a form that is immune to the MO,
most commonly by injecting RNA at the one-cell stage. For MOs targeted against
the translation start site, this might be achieved by removing the 5′
untranslated region of the mRNA or by introducing silent mutations into the
coding region. Such a difficulty does not arise for MOs designed to inhibit
pre-mRNA splicing. As mentioned above, different MOs may have different
penetrances, and this also holds true for different mRNAs. Thus, when doing
rescue experiments, it is important to achieve appropriate levels of injected
MO and mRNA. We therefore recommend paying particular attention to injection
volumes, injecting a variety of concentrations and also comparing embryos from
the same batch that have been injected with MO alone or with MO plus mRNA
(Little and Mullins,
2004).

For those genes that are expressed ubiquitously or that have no
overexpression phenotype, rescue experiments of this sort might be
straightforward. However, for genes that are expressed in a restricted manner,
or that have a strong overexpression phenotype, obtaining a true rescue may be
difficult or impossible. In particular, it might become necessary to drive
expression of the gene in its correct spatial pattern at the correct levels.
This may not be straightforward or even possible in some cases, but it might
be possible to devise molecular assays that suffice. For example, perfect
rescue of the loss of a mesoderm-inducing factor may not be possible because
the inducing factor has to be expressed in a precise spatiotemporal manner
(Piepenburg et al., 2004). It
might, however, be feasible to assay for the expression of one or more of its
target genes.

Conclusions

It should be apparent from this article that morpholino technologies
provide powerful approaches for the study of early development. Indeed, in
many cases, if one wants to know the function of a gene, currently there is no
alternative. But it should be equally apparent that interpretation of the
results of morpholino experiments, as for experiments in general, depends
entirely on appropriate controls. These controls allow the experimenter to
rule out off-target effects, and provide a high level of confidence in the
specificity of the MO and the validity of the results. As enterprising
researchers design more and more sophisticated uses for MOs, it behoves us all
to ensure that they are matched by the sophistication of the controls.
Therefore, our strongest recommendation is to take these controls seriously,
both as experimenters and as reviewers.

Acknowledgments

We thank our many colleagues, too numerous to name, for the technical
advances and lively discussions that have prompted us to write this review. We
apologise to the many colleagues whose work has not been cited. We
particularly thank John Postlethwait, Monte Westerfield, Kevin Dingwell and
Amer Rana for comments on earlier versions of the manuscript, and Chi-Bin
Chien and Richard Dorsky and the other reviewers of the manuscript for their
very helpful comments. Work in the Eisen Laboratory is supported by the
National Institutes of Health, the Broad Foundation and the Oregon Medical
Research Foundation. Work in the Smith Laboratory is supported by the Wellcome
Trust, the Biotechnology and Biological Sciences Research Council, and the
European Union Network of Excellence consortium `Cells into Organs'.

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